Plant Systematics and Evolution (2018) 304:343–355 https://doi.org/10.1007/s00606-017-1480-1

ORIGINAL ARTICLE

Unusual diversity of apomictic mechanisms in a species of ,

Ana Paula S. Caetano1,2 · Priscila A. Cortez3 · Simone P. Teixeira4 · Paulo E. Oliveira5 · Sandra M. Carmello‑Guerreiro6

Received: 4 July 2017 / Accepted: 7 November 2017 / Published online: 5 December 2017 © Springer-Verlag GmbH Austria, part of Springer Nature 2017

Abstract Apomixis, the asexual formation of seeds, seems to be a reproductive alternative for many angiosperms, involving various pathways with diferent genetic and ecological consequences. It is common in some megadiverse tropical groups such as Melastomataceae, of which approximately 70% of the species studied so far in the tribe Miconieae are autonomous apom‑ ictics. Hence, Miconia appears to be a good model for the study of the embryological pathways associated with apomixis. In the present study, we analyzed the polyploid and autonomous apomictic M. fallax and compared its embryology to that of the diploid and sexual M. pepericarpa, both treelets species common in the Cerrado, the Neotropical savanna areas of Central Brazil. Ovule structure and basic megasporogenesis and megagametogenesis events were similar in both species. However, M. fallax showed exclusive features associated with apomixis: aposporous embryo sac development, with the par‑ thenogenetic development of unreduced egg cells, autonomous endosperm formation, nucellar embryony and polyembryony. Moreover, both gametophytic and sporophytic apomixis occurred in parallel to the development of a sexual embryo sac, a rarely described condition, which probably confers a great reproductive fexibility to the species.

Keywords Adventitious embryony · Apospory · Autonomous apomixis · Megagametogenesis · Megasporogenesis · Polyembryony

Introduction

Handling editor: Hans de Jong. Despite the prevalence of sexual reproduction, apomixis— * Ana Paula S. Caetano the asexual production of seeds—is an alternative that seems [email protected] to have been important for the evolution and diversifcation of the angiosperms (Asker and Jerling 1992; Hojsgaard et al. 1 Programa de Pós‑Graduação em Ecologia e Conservação 2014). In general, the apomictic process bypasses important de Recursos Naturais, Instituto de Biologia, Universidade Federal de Uberlândia, Uberlândia, Minas Gerais, Brazil steps of sexual reproduction, notably both meiosis and egg cell fertilization, and has been viewed as a temporal and 2 Programa de Pós‑Graduação em Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, spatial deregulation of the sexual developmental program São Paulo, Brazil (Koltunow 1993; Koltunow and Grossniklaus 2003; Hand 3 Centro de Microscopia Eletrônica, Departamento de Ciências and Koltunow 2014). Currently, apomixis is considered an Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, important reproductive strategy often associated with geo‑ Bahia, Brazil graphical parthenogenesis and polyploidy (Carman 1997; 4 Departamento de Ciências Farmacêuticas, Faculdade de Hörandl et al. 2008; Santos et al. 2012). Ciências Farmacêuticas de Ribeirão Preto, Universidade de The seeds that result from the apomictic process can have São Paulo, Ribeirão Preto, São Paulo, Brazil embryos originating from a somatic cell of the ovule (sporo‑ 5 Instituto de Biologia, Universidade Federal de Uberlândia, phytic apomixis or adventitious embryony) or from an unfer‑ Uberlândia, Minas Gerais, Brazil tilized egg cell of an unreduced embryo sac (gametophytic 6 Departamento de Biologia Vegetal, Instituto de Biologia, apomixis). In the latter case, the unreduced embryo sac orig‑ Universidade Estadual de Campinas, Campinas, São Paulo, inates from a megaspore mother cell in which meiosis was Brazil

Vol.:(0123456789)1 3 344 A. P. S. Caetano et al. suppressed or altered (diplospory), or from a nucellar cell species are concentrated mainly on herbaceous Asteraceae called aposporous initial (apospory), by mitotic divisions in from temperate regions (Koltunow et al. 1998, 2000; Van both cases (Asker and Jerling 1992; Crane 2001; Hand and Dijk 2003; Noyes 2007), members of the tropical and mostly Koltunow 2014). woody Miconieae represent alternative models for studies of More than one somatic cell in an ovule giving rise to autonomous apomixis. embryos is a common phenomenon in adventitious embry‑ Despite their pollinator independence, some apomic‑ ony (Naumova 1993; Batygina and Vinogradova 2007). tic Miconieae species may produce viable pollen (Caetano Moreover, the successful embryogenesis in an ovule with et al. 2013b; Maia et al. 2016). Because these species receive multiple embryo sacs, either aposporous embryo sacs or visits from pollinators (Maia et al. 2016), we hypothesized aposporous and sexual embryo sacs, can lead to polyem‑ that the sexual mechanism would co-occur with the apom‑ bryony. Therefore, the sexual and asexual processes may ictic one. Secondly, we hypothesized that pollination-inde‑ occur in the same or in diferent seeds in a given apomictic pendent species could produce an autonomous endosperm. species (Asker and Jerling 1992; Koltunow and Grossniklaus In this study, we show similar sexual events of megasporo‑ 2003; Batygina and Vinogradova 2007). Indeed, most genesis and megagametogenesis in the apomictic and polli‑ are facultative apomictic. From an evolutionary perspective, nation-independent M. fallax DC. and its congener M. pep- the facultative apomixis seems to be advantageous by keep‑ ericarpa Mart. ex DC. Furthermore, M. fallax produces an ing both asexual and sexual reproductive modes (Koltunow autonomous endosperm, and the co-occurrence of apospory, and Grossniklaus 2003; Hojsgaard and Hörandl 2015). On adventitious embryony and sexuality, leads to development the other hand, the apomixis is the only mode of reproduc‑ of polyembryonic seeds, showing a mixed reproductive strat‑ tion by seeds in obligate apomictic species (Koltunow 1993; egy in this markedly diverse group. Richards 1997), which have been pointed out as exceptions in natural populations (Nogler 1984; Asker and Jerling 1992; Hojsgaard and Hörandl 2015). Materials and methods Although apomixis usually bypasses sexual reproduction stages, not all apomicts are completely pollination independ‑ materials ent. Most species are pseudogamous, which means that the nuclear fusion between the male gamete and the central cell The two species of Miconia studied here are common shrubs/ is required for functional endosperm development. Pseudog‑ trees in the Cerrado, the Neotropical savanna region of Cen‑ amous apomicts seem to be prevalent among species with tral Brazil, and have been previously studied for breeding adventitious embryony or apospory. Pseudogamy is unneces‑ system features by Goldenberg and Shepherd (1998). On sary only in autonomous apomicts, an apparently uncommon the one hand, the pollination-independent, apomictic and condition, prevalent among diplosporous apomicts (Asker polyploid M. fallax is a shrub species with a wide distri‑ and Jerling 1992; Koltunow and Grossniklaus 2003; Whit‑ bution from Peru, Venezuela and Guiana to southeastern ton et al. 2008). Brazil. On the other hand, the diploid and sexual species M. Sporophytic and/or gametophytic apomixis has been pepericarpa is a Cerrado tree with a distribution restricted reported for 78 fowering plant families and has arisen sev‑ to some Brazilian states (Goldenberg and Shepherd 1998; eral times during the evolutionary history of angiosperms Goldenberg 2009; Caetano et al. 2013b). (Hojsgaard et al. 2014). Asteraceae, Poaceae and Rosaceae The samples were collected in Cerrado fragments of are historically cited as containing the majority of known Itirapina, São Paulo State, Brazil (lat. 22°15′10″S, long. apomictic genera (Carman 1997; Hojsgaard et al. 2014). 47°49′22″W). Voucher specimens were deposited in the However, the mostly tropical Melastomataceae has gained Herbarium of Universidade Estadual de Campinas (UEC), prominence due to their high frequency of autonomous São Paulo State, Brazil, under the numbers 150450 (M. fal- apomictic species, particularly within the tribe Miconieae, lax) and 150451 (M. pepericarpa). which contains up to 70% of apomictics species (Renner 1989; Goldenberg and Shepherd 1998; Goldenberg and Var‑ Megasporogenesis, megagametogenesis, assin 2001; Santos et al. 2012; Maia et al. 2016). and endosperm and apomictic embryo origin Studies concerning the cytological mechanisms of apomixis and their relation with sexuality within Mico‑ Megasporogenesis and megagametogenesis analyses for both nieae have revealed important insights that are useful for species were carried out using diferent developmental stages developmental, taxonomic and ecological knowledge, but from fower bud to fower anthesis. The origin of apomictic still involve a small percentage of species in this megad‑ embryos was determined also using seeds of M. fallax at iverse group (Borges 1991; Cortez et al. 2012; Caetano several developmental stages. Buds, fowers and fruits were et al. 2013a, b). Since studies with autonomous apomictic collected from six individuals per species. Finally, the origin

1 3 Sexual and apomictic development in Miconia, Melastomataceae 345 and development of autonomous endosperm in M. fallax was shared conditions, we observed some cytological difer‑ determined from three individuals using seeds obtained from ences, related to the reproductive system. fruits of previously emasculated and bagged fower buds. All samples were fxed in 2% glutaraldehyde, 4% formal‑ Ovule structure dehyde and 0.1 M phosphate bufer, pH 6.8 (McDowell and Trump 1976) for 24 h, and then dehydrated through a graded We observed that the ovules are anatropous (M. fallax) ethanol series, embedded in methacrylate resin (Historesin or hemi-anatropous (M. pepericarpa), crassinucellate Leica), and sectioned with a tungsten carbide knife on a rota‑ and bitegmic, with a “zigzag” micropyle (Fig. 1a–e). The tory microtome (RM 2245, Leica Microsystems). Transverse outer integument is three-layered and the inner one is bi- and longitudinal serial sections, between 1 and 5 μm thick, layered, both being non-vascularized (Fig. 1a–c). Pericli‑ were stained with 0.05% toluidine blue (CI 52040) in citrate nal and oblique cell divisions can occur in the micropylar bufer, pH 4.8 (modifed from O’Brien et al. 1964). Obser‑ region of the outer integument, making it thicker (Fig. 1d). vations were made and photographs were obtained using The raphe has one vascular bundle extending from the an Olympus BX51 microscope equipped with an Olympus funicle to the chalaza, where compact cells with a dense DP71 digital camera. cytoplasm and prominent nucleus form a non-lignifed hypostase (Fig. 1a, b, 3e). In M. pepericarpa the vascular Pollen tube growth bundle is surrounded by cells containing phenolic com‑ pounds (Fig. 1c). Stigmatic pollen deposition and pollen tube growth were M. fallax analyzed as an indicator of sexual reproduction in . Megasporogenesis and megagametogenesis Senescent fowers that had lost petals and stamens, but with an intact gynoecium, were fxed in formalin–acetic acid–eth‑ Although structural diferences between M. fallax and anol (FAA) solution (1:1:18; v/v/v) (Johansen 1940). Styles M. pepericarpa were observed from the initial stages of were then cleared and softened in 7 N sodium hydroxide megasporogenesis onwards, sexual events represented by (NaOH) at room temperature for 30 min, washed with water megaspore mother cell diferentiation, meiosis and mega‑ and stained with water-soluble 0.1% aniline blue prepared spores formation, and embryo sac development, occurred in 0.1 N K­ PO . Observations were made, and photographs 3 4 in a similar way in both species (Figs. 2, 3). were taken under UV light (Martin 1959) using an Olympus In M. fallax and M. pepericarpa, the megaspore mother BX51 microscope equipped with an Olympus DP71 digital cell enlarged within the nucellus (Fig. 2a–c) and, through camera. meiosis, gave rise sequentially to a dyad (Fig. 2d) and to a linear tetrad of megaspores (Fig. 2e, f). The functional Polyembryony analysis megaspore was chalazal (Fig. 2e, f) and, after three rounds of mitotic divisions (Fig. 3a–c), originated an embryo sac The presence and frequency of polyembryony in M. fallax of the Polygonum-type, with three antipodals, one binu‑ were determined using 1200 seeds obtained from mature cleate central cell (Fig. 3d), two synergids (Fig. 3e) and fruits from six individuals. The seeds were removed from the one egg cell (Fig. 3e, f). In M. pepericarpa, fertilization fruits, immediately immersed in 10% sodium hypochlorite was demonstrated by one pollen tube penetrating a syn‑ (NaClO) solution for 3 min, washed in distilled water and ergid, with the subsequent formation of a zygote and a distributed inside plastic boxes with wet flter paper over‑ nuclear endosperm (Fig. 3g, h). Although direct evidence lying cotton wool. The seed germination experiment was of fertilization was not detected for M. fallax, we observed conducted under continuous white fuorescent light and con‑ pollen grains germinating on the stigmatic surface, and trolled temperature of 28 °C. Germinated seeds were dis‑ pollen tubes growing through the styles of senescent fow‑ sected under a stereomicroscope for both seedling counting ers (Fig. 3i, j). and morphological analyses. For M. fallax, 69.05% of the 42 ovules observed had aposporous initial cells around the megaspore mother cell (Fig. 4a), megaspores (Fig. 4b, c) or developing embryo Results sac (Fig. 4d). The aposporous initial cells were similar to the functional megaspore, with a dense cytoplasm, conspicuous central nucleus, and one or two vacuoles Miconia fallax and M. pepericarpa were similar in terms of (Fig. 4a–d). These aposporous initials gave rise to one to ovule structure and some events of megasporogenesis and three aposporous embryo sacs, structurally similar to the megagametogenesis as detailed below. Apart from these Polygonum-type (Fig. 4e, f). Ovules in fresh open fowers

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Fig. 1 Ovule structure in sexual and apomictic species of Miconia. a, b Longitudinal sec‑ tions of ovules in M. fallax and M. pepericarpa, respectively, demonstrating anatropous (a), hemi-anatropous (b) and biteg‑ mic ovules, with a single vas‑ cular bundle and a hypostase. c Transverse section showing a three-layered outer integument and a bi-layered inner integu‑ ment in M. pepericarpa. Arrows indicate phenolic-storing cells around the vascular bundle. d “Zigzag” micropyle in the M. fallax ovule in longitudinal sec‑ tion, also demonstrating pericli‑ nal divisions forming additional layers in the outer integument (asterisk). e Detail of hypostase cells in the M. fallax ovule. F funicle, H hypostase, II inner integument, OI outer integu‑ ment, M micropyle, VB vascular bundle

generally contained one mature embryo sac, while the oth‑ In seeds from immature fruits, the endosperm was at the ers were under development. nuclear developmental stage inside the embryo sac, includ‑ ing those obtained from emasculated fower buds (Fig. 4l, m). The presence of more than one embryo per seed con‑ Embryo and endosperm origin in Miconia fallax frmed that the species is polyembryonic (Fig. 4n), but it was not possible to distinguish between possibly zygotic The embryos of M. fallax developed from each egg cell of and apomictic embryos. The events of sexual and apomic‑ one or more embryo sacs of the same ovule, as demonstrated tic reproduction in M. fallax can be viewed in the drawing by the embryo position inside the embryo sac (Fig. 4g–i). of Fig. 5 in a summary form. Moreover, in ovules from the same fower, supernumerary embryos could develop from nucellar cells that were posi‑ tioned next to the hypostase (Fig. 4j) or around the embryo sac (Fig. 4k, l).

1 3 Sexual and apomictic development in Miconia, Melastomataceae 347

Fig. 2 Megasporogenesis in sexual and apomictic species of Mico- (arrowhead). e, f Megaspore tetrad in M. fallax and M. pepericarpa, nia. a Longitudinal section of a M. fallax ovule, showing a megaspore respectively, with the functional chalazal megaspore. FM functional mother cell (MMC) in the nucellus. b, c Details of MMC in M. pep- megaspore, MD megaspore dyad, MMC megaspore mother cell, MT ericarpa and M. fallax, respectively. d Megaspore dyad in M. fallax, megaspore tetrad in which the calazal one goes through a second meiotic telophase

Polyembryony in Miconia fallax that were markedly different in size and morphology (Fig. 7f–h). The smaller seedlings often had poorly devel‑ Miconia fallax seed germination occurred seven days after oped and morphologically irregular cotyledons (Fig. 7g, h). the beginning of the experiment. The mean percentage of In some seeds (2.9%) the cotyledons were oriented toward germinated seeds was 67.4% (± 19.1). Polyembryony was the micropylar region (Fig. 7b). These inverted seedlings confrmed in the studied population, with a frequency of were originated from monoembryonic as well as polyem‑ 34.2%. The maximum number of seedlings in a seed was bryonic seeds. four, with a mean of 1.4 (± 0.1) seedlings per seed. Seeds containing one seedling were the most frequent case, cor‑ responding to 66.1% of the germinated seeds, followed by Discussion those with two (28.49%), three (4.55%) and four seedlings (0.85%) (Figs. 6, 7a–h). Although the ovule structure and basic megasporogenesis More than half of the seeds with two seedlings (50.4%) and megagametogenesis events were similar for M. fallax and all seeds with three or four seedlings exhibited seedlings and M. pepericarpa, in the apomictic M. fallax, we observed

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Fig. 3 Megagametogenesis in sexual and apomictic species of Mico- M. fallax. g Pollen tube reaching the embryo sac (arrow) in M. pep- nia. a Miconia fallax: binucleate (arrows) embryo sac. b Binucle‑ ericarpa. h Detail of zygote and an endosperm nucleus in M. pep- ate embryo sac with the micropylar nucleus at mitotic anaphase. c ericarpa. i, j Pollen grains germinating on the stigma surface (i) Tetranucleate (arrows) embryo sac. d, e Details of the mature embryo and pollen tubes growing in style (j) of M. fallax. EC egg cell, EN sac showing polar nuclei (d), synergids and egg cell (e) in M. pep- endosperm nucleus, PN polar nuclei, S synergid, Z zygote ericarpa. f Detail of the mature embryo sac showing an egg cell in aposporous embryo sacs development, with the later parthe‑ and the “zigzag” micropyle are stable conditions within Mel‑ nogenetic development of unreduced egg cells, autonomous astomataceae (Tobe and Raven 1983; Medeiros and Mor‑ endosperm formation and nucellar embryony. Our results retes 1996; Cortez and Carmello-Guerreiro 2008; Caetano indicate that this species is autonomous apomictic, with both et al. 2013a; Ribeiro et al. 2015; Caetano et al. 2017). How‑ gametophytic (apospory) and sporophytic apomixis. Moreo‑ ever, the outer integument thickness and the ovule curvature ver, we suggest that M. fallax is a facultatively apomictic are variable characters within the family (Tobe and Raven species, exhibiting a mixed reproductive system in which 1983; Medeiros and Morretes 1996; Cortez and Carmello- the apomixis occurs in parallel to sexuality. Guerreiro 2008; Caetano et al. 2013a; Ribeiro et al. 2015; Caetano et al. 2017). Since embryological characters are Ovule structure recognized due to their systematic importance (Davis 1966; Tobe 1989; Johri et al. 1992), and the outer integument The structural characteristics of the M. fallax and M. pep- thickness has been useful for clade delimitation within Mel‑ ericarpa ovules were similar. The crassinucellate ovule with astomataceae (Caetano et al. 2017), the systematic signif‑ two not vascularized integuments, the inner one two-layered, cance of ovule curvature is yet to be tested for the group.

1 3 Sexual and apomictic development in Miconia, Melastomataceae 349

Fig. 4 Megagametogenesis and embryogenesis in the apomictic spe‑ sac. h, i Developing embryos (apomictic and/or sexual), indicated by cies Miconia fallax. a–d Aposporous initial cells, indicated by arrow‑ arrows and probably originated from egg cells, within embryo sacs heads, diferentiate above the megaspore mother cell (arrow) (a), meg‑ (triangles). j Adventitious proembryo, indicated by an arrow. k–m aspore tetrad (b), functional megaspore (arrow) (c) and developing Adventitious embryos, indicated by arrows (k, l) and autonomous embryo sacs (triangles) (d). e Ovule with a tetranucleate and binucle‑ endosperm development, indicated by arrows head (l, m). n Polyem‑ ate embryo sacs (triangles). Arrows indicate the embryo sacs nuclei. bryonic seed with three visible embryos (triangles). A Antipodals, AIC f Ovule with a mature and two developing embryo sacs (triangles). aposporous initial cell, EC egg cell, FM functional megaspore, MMC g Developing proembryo, indicated by an arrow, within an embryo megaspore mother cell, MT megaspore tetrad, PN polar nuclei

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Fig. 5 Events of sexual and apomictic reproduction in the ovule of (blue). e Octanucleate (red) and tetranucleate (blue) embryo sacs. f Miconia fallax. Cells and structures involved in the sexual processes Pollen tube reaching the mature embryo sac (red) and octanucleate are shown in red, while those associated with apomixis are displayed embryo sac (blue). g Zygotic proembryo and endosperm (red) and in blue and yellow. a, b Diferentiated megaspore mother cells (red) mature embryo sac (blue). h, i Zygotic embryo and endosperm (red), in the nucellus. c Megaspore tetrad (red) and aposporous initial cell aposporous proembryo and autonomous endosperm (blue) and adven‑ (blue). d Binucleate embryo sac (red) and aposporous initial cell titious proembryos (yellow)

Megasporogenesis, megagametogenesis 1995; Koltunow et al. 1998; Naumova and Vielle-Calzada and the structural basis of apomixis 2001; Guan et al. 2006; Galla et al. 2011). Apparently, a correct positioning of AICs in the ovule is important for the Miconia fallax and M. pepericarpa exhibited a similar sex‑ development of the aposporous embryo sac, probably due ual development process, indicating that apomixis does not to the expression of particular factors restricted to a specifc alter completely the phylogenetically conservative events of area in the ovule (Koltunow et al. 1998; Galla et al. 2011). megasporogenesis and megagametogenesis in the apomictic Moreover, a cellular polarization resulting from vacuole M. fallax species. The linear megaspore tetrad formation, development seems to mark the transition of the aposporous with the functional chalazal megaspore, and Polygonum- initial cells to the aposporous embryo sac (Naumova 1997; type embryo sac are conditions that seem to be stable within Naumova and Vielle-Calzada 2001). Melastomataceae (Tobe and Raven 1983; Johri et al. 1992; The diferentiation and development of more than one Medeiros and Morretes 1996; Caetano et al. 2013a) and AIC gave origin to multiple embryo sacs in a single ovule possibly common among sexual and facultative apomictic of M. fallax. In fact, several AICs may develop within the species. nucellar tissue, giving rise to ovules with supernumerary Despite these similarities, M. fallax exhibited embryo‑ embryo sacs (Koltunow et al. 1998; Albertini et al. 2001; logical diferences related to apomixis. The aposporous ini‑ Koltunow and Grossniklaus 2003; Guan et al. 2006; Yao tial cells (AICs) started to develop concomitantly with the et al. 2007). This indicates that in aposporic species, all megasporogenesis and were identifed by their position, as nucellar cells carry the genetic information needed to well as by structural features (see Naumova and Willemse develop into embryo sacs, even if only a few cells, adjacent

1 3 Sexual and apomictic development in Miconia, Melastomataceae 351

to the embryo sac and/or megaspores, actually do so (Nau‑ mova 2008). On the other hand, the occurrence in a sin‑ gle ovule of both aposporous and sexual embryo sacs, as observed in M. fallax, does not appear to be a common event (Nogler 1984; Alves et al. 2001). The apospory frequently leads to the abortion of the sexual process (Koltunow et al. 1998; Wen et al. 1998; Tucker et al. 2003; Yao et al. 2007), suppressing this event through changes in gene expression (Tucker et al. 2003), which did not appear to occur in the species studied here. Since our results demonstrated the capacity of reduced embryo sac formation and pollination, and pollen viability was about 29% (Caetano et al. 2013b), we suggest that M. fallax is a facultative apomictic species. It has been pro‑ posed that since the apomictic representatives of Melasto‑ mataceae with relatively high pollen viability attract pol‑ linators, they might produce seeds through both apomictic and sexual events (Maia et al. 2016). In fact, the majority of the apomictic plants retain the ability to reproduce sexu‑ Fig. 6 Percentage of germinated seeds with one, two, three or four ally, and some degree of sexuality has been associated with Miconia fallax seedlings in the apomictic species the three kinds of apomictic mechanisms (Bicknell et al. 2003; Martins and Oliveira 2003; Aliyu et al. 2010; Sartor et al. 2011; Noyes and Givens 2013). Apparently, the ratio

Fig. 7 Seedling emergence and morphology in the apomic‑ tic species Miconia fallax. a–c Seedling emergence from monoembryonic (a) and poly‑ embryonic (b, c) seeds. Arrows indicate embryos. In fgure b it is possible to see one embryo with the cotyledons oriented to the micropylar region (arrow‑ head) and other embryo with a root apex oriented to the micro‑ pylar region (arrow). d Seedling from a monoembryonic seed. e Morphologically similar seed‑ lings from a polyembryonic seed. f–h Morphologically dif‑ ferent seedlings from seeds with two, three and four embryos, respectively

1 3 352 A. P. S. Caetano et al. between sexual and asexual reproduction in some apomic‑ 1944), indicating that polyembryony is not always related to tic species may vary under diferent environmental condi‑ apomixis in this group. tions, since diferent stress situations alter the proportion The frequency of polyembryonic seeds in Melastoma‑ of sexual embryo sacs in facultative apomictic Eragrostis taceae varies from 0.18 to 34.2% and depends on both the curvula (Rodrigo et al. 2017). The importance of sexuality species and population analyzed (Mendes-Rodrigues and in facultative apomictics is the possibility of maintenance of Oliveira 2012). Although up to fve embryos may occur some genetic diversity within the species, conferring adapt‑ in a single Melastomataceae seed, the mean number of ability to this taxon (Mazzucato et al. 1996; Richards 1997; embryos per seed is always close to one (Mendes-Rodrigues Bicknell and Koltunow 2004). Moreover, sexuality could and Oliveira 2012). A tendency to a reduction of embryo avoid genomic decay and extinction in facultative apomictic size and to an increase in the seedling mortality ratio has taxa (Hojsgaard and Hörandl 2015). been suggested for seeds with a larger number of embryos The formation of an embryo by parthenogenesis of the (Ladd and Cappuccino 2005; Mendes-Rodrigues et al. 2011, egg cell of an aposporous embryo sac in M. fallax allows 2012), indicating a selective pressure to reduce the num‑ classifying this species as having gametophytic apomixis of ber of embryos per seed. It is possible that the seed size, the apospory type. At the same time, M. fallax also exhib‑ particularly regarding the small seeds of Melastomataceae, its sporophytic apomixis, demonstrated by the development restricts the proper development of multiple embryos. This of the adventitious nucellar embryos. The presence of two possibility is supported by the diferent sizes and morpho‑ apomictic mechanisms in a single Miconieae species cor‑ logical anomalies among seedlings from polyembryonic roborates the idea that sporophytic and gametophytic apo‑ seeds observed here for M. fallax, for other Melastomataceae mixis can both occurs in parallel (Naumova 1993; Koltunow (Mendes-Rodrigues and Oliveira 2012) and for other poly‑ and Grossniklaus 2003; Naumova 2008; Yao et al. 2007). embryonic species (Koltunow et al. 1998; Mendes-da-Glória Furthermore, the occurrence of diplospory in M. albicans et al. 2001; Costa et al. 2004; Bittencourt and Semir 2005; (Caetano et al. 2013a) makes the genus a repository of all Mendes-Rodrigues et al. 2005, 2012). types of apomictic pathways described to date and suggests Despite these possible size restrictions, some advantages that their regulation may not be that diferent (Rutishauser have been attributed to polyembryony such as the increased 1982). chance of occurrence of at least one established seedling per Miconia fallax also exhibited an endosperm formation seed (Ladd and Cappuccino 2005; Hotchkiss et al. 2008) without fertilization. In the apomictic species with autono‑ and the increased ftness of individuals growing in patches mous endosperm development such as M. fallax (this study), (Allee efect) (Cappuccino 2004). In this respect, it is sug‑ M. albicans (Caetano et al. 2013a), Hieracium (Koltunow gested that polyembryony, apparently a frequent processes et al. 1998) and Taraxacum (Cooper and Brink 1949), in Miconieae, can favor both the reproductive success and there is a tolerance of unbalanced maternal versus paternal frequency of this tribe in the Neotropics (Mendes-Rodrigues genome contributions (m:p), because there is no paternal and Oliveira 2012). contribution to endosperm formation. Strategies that aim to tolerate the unbalance in the m:p ratio, or ensure the 2m:1p ratio, have been interpreted as prerequisites for apomixis Conclusion maintenance (Grimanelli et al. 1997; Quarin 1999; Kol‑ tunow and Grossniklaus 2003) and could explain, among The polyploid M. fallax showed some exclusive features other factors, the frequency of this reproductive system in associated with apomixis, such as aposporous initial cell dif‑ Melastomataceae. ferentiation, more than one embryo sac in a single ovule, embryo development by apospory and adventitious embry‑ ony, autonomous endosperm formation and polyembryony. Polyembryony in Miconia fallax But it retains the mechanisms for reduced embryo sac pro‑ duction and sexual reproduction observed in the studied pop‑ The development of aposporous and adventitious apomic‑ ulation. These distinct mechanisms provide great reproduc‑ tic embryos, associated or not with the zygotic ones, led to tive fexibility to the species, associating independence of polyembryony in M. fallax. On the other hand, monoem‑ pollinators with eventual sexual embryo production, bring‑ bryony was reported in M. pepericarpa and other sexual spe‑ ing together gene variability and reproductive assurance. In cies of Melastomataceae (Mendes-Rodrigues and Oliveira addition, the occurrence of diplosporous apomixis elsewhere 2012). The association between apomixis and polyembry‑ in the genus makes the Miconia a repository of all apomixis ony has been observed for diferent Melastomataceae spe‑ pathways recorded to date. It has been proposed that apo‑ cies (Mendes-Rodrigues and Oliveira 2012), but multiple mixis may enhance the diversifcation rates of some plant embryos can originate from suspensor cells (Subramanyan groups, and the occurrence of this diversity of reproductive

1 3 Sexual and apomictic development in Miconia, Melastomataceae 353 alternatives in the Miconia may explain, among other fac‑ Cooper DC, Brink RA (1949) The endosperm-embryo relationship Taraxacum officinale tors, its enormous species richness. in an autonomous apomict, . Bot Gaz 111:139–153. https://doi.org/10.1086/335582 Acknowledgements Cortez PA, Carmello-Guerreiro SM (2008) Ontogeny and structure This work is part of the Master’s dissertation of the pericarp and the seed coat of Miconia albicans (Sw.) Tri‑ of the frst author. We thank the São Paulo Research Foundation ana (Melastomataceae) from “cerrado”Brazil. Revista Brasil Bot (FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo) for 31:71–79. https://doi.org/10.1590/S0100-84042008000100008 the fnancial support (Grant Numbers 2008/10793-0 and 2010/15077-0) Cortez PA, Carmello-Guerreiro SM, Teixeira SP (2012) Under‑ and J.Y. Tamashiro for help with the feldwork. 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